Fig. 6 Protein sequence comparison with clustAl alignment reveals highly conserved features of E. coliBacA and mycobacterial Upk. Identity of E. colito M. smegmatis and M. tuberculosis is 38 % and similarity 50 %(A). Identity of M. smegmatis to M. tuberculosis is 73 %, and similarity 78 %(B). Color definitions: blue - all amino acids of a column are identical; red - more than half of the amino acids of a column are identical or belong to one of the strong groups (amino acids with strong similarities); orange - more than half of the amino acids of a column belong to one of the weak groups (amino acids with weak similarities), or amino acids that could be grouped into a weak group with every amino acid of the same column belonging to a strong group that is marked red.

An in-frame, unmarked deletion of the M. smegmatisupk gene was created by a two-step-method [58]. Regions of 879 bp and 945 bp flanking the upk gene in M. smegmatis were amplified by PCR and inserted into pYUB657. Thus 96% of the wildtype upk gene was deleted. The mutated copy of upk was introduced into M. smegmatis by electroporation. HygR transformants which displayed a SucS phenotype were assayed for recombination by Southern blot. There is the possibility of integration upstream or downstream of the target gene by single crossover. A mutant in which the template plasmid has inserted upstream, resulting in 11,547 bp and 7,337 bp fragments on Southern blot (Fig 7., southern blot a) was chosen for further analysis. A culture of this single crossover strain was grown overnight in complete medium without hygromycin. This period of growth allowed double crossover events to occur, followed by plasmid loss. Deletion or preservation of the wildtype allele occur with equal probability. Two out of 6 analyzed clones displayed the characteristics of a upk deletion mutant (Fig. 7, southern blot b). The mutant strain was named M. smegmatisΔupk. A complementation strain was constructed, using pMV262-rv2136c, a multicopy plasmid expressing the M. tuberculosis H37Rv homologue of the upkgene under control of the M. bovis BCG groEL2 (Hsp60) promoter. The parent plasmid, pMV262, is an episomal E. coli-mycobacteria shuttle plasmid.

Fig. 7 An in-frame, unmarked deletion of upk was generated in M. smegmatis by a two step approach. In the first step, the counterselectable suicide plasmid pYUB657 carrying the deletion allele of upkrecombined with the bacterial chromosome. Southern blot analysis confirmed the recombination event. Two orientations are possible (southern blot a: lane 1 and 2). Clone 2 was selected. In the second step the plasmid loops out in the absence of hygromycin selective pressure. The deletion allele or the wildtype copy of upkare lost with equal probability. Deletion of upk was verified by Southern blot (Southern blot b). Lane 1 and 5 are deletions, the others are wildtype. For the following studies clone number 1 was selected.

Bacteria were grown on 7H10 agar plates. While wildtype M. smegmatis showed dome-like morphology, Δupk mutant colonies exhibited caved-in structures (Fig. 8). Although this observation suggests that the cell wall is affected, examination by electron microscopy revealed no differences (Fig. 8). Yet, specific immunogold-staining of peptidoglycan revealed a lower number of gold-particles associated with the cell surface of the Δupkmutant (Table 1). Twenty electron microscopy images of stainings of wildtype and the Δupk mutant were examined. For wildtype, 241 particles were counted of which 27% were surface associated. Of the 225 particles counted on the Δupk mutant bacteria, only 13% were surface associated (Fig. 9).

Fig. 9 Distribution of gold particles in an anti-peptidoglycan immuno-gold stain. Twenty electron microscopy images of wildtype and the Δupk mutant were examined. For wildtype, 241 particles were counted of which 27% were surface associated. Of the 225 particles counted on the Δupk mutant bacteria, 13% were surface associated.

As described above, Δupk mutants are expected to be more sensitive to bacitracin than wildtypeM. smegmatis. This issue was investigated by the alamar blue assay. Alamar blue is a redox-dye that converts from blue to red upon reduction, reflecting a measure of growth [59]. Bacteria were incubated over night in 7H9 complete medium supplemented with serial dilutions of bacitracin (0 U/ml – 2,500 U/ml). Next day, alamar blue was added and fluorescence at 570 nm was measured. The M. smegmatisΔupk mutant showed no growth at a concentration of 500 U/ml bacitracin, whereas wildtype reached 55% of its maximal growth (Fig. 10). The reconstituted strain M. smegmatisΔupk + pMV262-rv2136c failed to regain wildtype growth but displayed an intermediate phenotype.

Fig. 10 Sensitivity assay to bacitracin. M. smegmatis mc2155 (■), M. smegmatis mc2155Δupk(□),and M. smegmatis mc2155Δupk+ pMV262-rv2136c (●) were incubated with different concentrations of bacitracin over night. Next day alamar blue was added and growth was measured at 570 nm. The Figure shows 1 representative experiment of 3 with similar results.

* Curves were significantly different at 500 and 1000 U bacitracin / ml according to Mann Whitney test (P<0.0001).

We wanted to find out whether upk homologues contribute to virulence of pathogenic mycobacteria. Therefore we chose the model of macrophage infection. Although, M. smegmatis fails to persist in macrophages it was possible to compare the survival times of M. smegmatis and the Δupk mutant in murine bone marrow derived macrophages. After infection, bacteria were cleared quickly. The time needed to kill half of the bacterial number upon infection differed between M. smegmatis wildtype (98 min), M. smegmatisΔupk mutant (27 min) and the reconstituted mutant strain (126 min) (Fig. 11).

Fig. 11 Infection of murine bone marrow derived macrophages. M. smegmatis mc2155 (■), M. smegmatis mc2155Δupk (□) and M. smegmatis mc2155Δupk+ pMV262-rv2136c (●) were used at a MOI of 200. After 27 min half of M. smegmatisΔupk mutants were killed. This was true for wildtype M. smegmatis after 98 min and after 126 min for Δupk mutant carrying pMV262-rv2136c. The Figure shows 1 representative experiment of 2 with similar results.

According to Mann Whitney test, growth curves in 7H9 complete medium (A) were not different whereas growth of M. smegmatis wildtype was significantly different at 9 and 24 h in biofilm medium(B) compared to the M. smegmatisΔupk strain and the reconstituted strain (P<0.0001).

Principally, an intact biofilm is characterized by a matrix consisting of extracellular polymer substances (EPS). Such a matrix was clearly visible for wildtype M. smegmatis, whereas the Δupkmutant grew in separated single cells (Fig. 13). Additional studies to follow in vivo[page 56↓]biofilm development were performed. M. smegmatis wildtype applied to a mouse penis induced smegma in 65 % of the animals, whereas smegma development upon application of Δupk deletion mutant was observed for 42% only, which was about background level (Fig. 14A). Groups consisted of 10 animals each.

Fig. 14A In vivo biofilm formation. Penises of C57BL/6 mice were exposed to medium, wildtype M. smegmatis, and Δupk deletion mutant. The Figure shows combined results of 2 independent experiments with similar results. Before application of the M. smegmatisstrains, mice had a clean penis (14B). A penis with an over night grown smegma is depicted in 14C. 14D shows a smegma plug removed from a penis.

The M. smegmatis upk gene was identified and an in-frame, unmarked deletion mutant was generated. The absence of Upk influenced bacitracin sensitivity, peptidoglycan synthesis, survival in macrophages, growth properties, and biofilm formation in vitro and in vivo. In vitro biofilms of M. smegmatisΔupk mutant were characterized as scattered islands of bacteria compared to a completely covered surface observed for wildtype bacteria. The M. smegmatisΔupk strain is the first to express a phenotype in a newly developed in vivo mouse model of biofilms.

A temperature sensitive (ts) TM4 phage delivery system was used to generate hygromycin-marked knockout mutants in M. tuberculosis and M. bovis BCG. Flanking regions to the M. tuberculosis upk gene were cloned next to a hygromycin resistance cassette and fused to the phage genome. Recombinant ts phages were amplified at 30°C using M. smegmatis growing in top agars as a host. At 37°C, phages were impaired to become lytic and could be used to transduce M. tuberculosis H37Rv and M. bovis BCG. The recombinant phages transferred their DNA into the bacteria. Flanking regions induced double crossover events, thus exchanging the upk gene with a hygromycin resistance cassette, which subsequently was verified by Southern blot (Fig. 15). The mutant strains were named: M. tuberculosisΔupk and M. bovis BCG Δupk.

Fig. 15 Construction of upk knockout mutants in M. tuberculosis and M. bovis BCG. Knockout phage genome phAE159-Δupk was generated by ligation of the plasmid pKO-upk and the phage genome phAE159 TM4 (A). Four phages were tested for temperature-sensitivity and revealed the expected phenotype- lysis at 30°C but not at 37°C (B). # 4 was picked, amplified and used for transduction of M. tuberculosis and M. bovis BCG. Lane 4 and 6 represent upk deletion mutants for M. tuberculosis H37Rv and M. bovis BCG, verified by Southern blot analysis (C).

A complementation strain of M. tuberculosis was constructed using pMV262-rv2136c, a multicopy plasmid expressing upkunder control of the groEL2 (Hsp60) promoter ofM. bovis. The parent plasmid, pMV262, is an episomal E. coli-mycobacteria shuttle-plasmid. M. [page 60↓]tuberculosis was difficult to transform by electroporation. In many cases, only the antibiotic resistance was transformed and other parts of the plasmid were lost. Thus, the appropriate size of the resistance cassette and the rv2136c gene (data not shown) was verified by PCR for kanamycin resistant colonies of M. tuberculosis H37Rv Δupk mutants, transformed with pMV262-rv2136c. Additionally, whole DNA was prepared from a positive clone and used for transformation ofE. coli DH5α. Subsequently, the plasmid was purified and compared to the original vector. BamHI digest, released the rv2136c insert. SalI digest was used to verify the correct orientation of the insert. The fragments were analyzed by gelelectrophoresis and appeared as the same size as the original vector. The recombinant M. tuberculosisΔupk knockout clone carrying this vector was used as the complementation strain (Fig. 16). Transcription levels of upk were determined by RT-PCR in relation to rv2946c, a gene transcribed at the same level inthe M. tuberculosis H37Rv Δupk mutant and wildtype. Transcription of the upk gene under control of the groEL2 promoter was 3.51 fold higher than rv2946c-transcription and 201.56 fold lower than transcription of upk in the wildtype strain under control of the natural promoter in late logarithmic growth phase.

Fig. 16 Comparison of the original reconstitution vector pMV262-rv2136c, and the reconstitution vector purified from an electroporated Δupk strain. Lanes # 1 and # 2 show the pattern of the original insert with accurate orientation. Lanes # 3 and # 4 confirm this pattern for the vector purified from the reconstitution strain.

Growth properties in 7H9 medium were examined for the 3 strains: wildtype, Δupk mutant, and reconstitution. OD600 was measured once a day. The M. tuberculosisΔupkmutant showed no differences in growth rate compared to wildtype whereas the complementation strain grew more slowly (Fig. 17).

Colonies of M. tuberculosis wildtype, the M. tuberculosisΔupk mutant and the reconstituted strain were of the same shape (data not shown). The attenuated strain M. tuberculosis H37Ra is known to behave differently compared to M. tuberculosis H37Rv in the following in vitro assays: Neutral red staining, cording assay, and pellicle culture. The H37Ra-strain is not stainable with neutral-red [61] (Fig. 18A). For virulent M. tuberculosis H37Rv, bacterial cord-factors e.g. trehalose dimycolate (TDM; [62] are necessary for growth in cords on a surface in non-shaking cultures [63]. After 2 to 3 weeks, cultures of M. tuberculosis grown without shaking in Sauton medium, form pellicles on top of the medium and bacteria push each other up the wall of the tube.

Also cording was not affected by loss of upk. All strains were stained with rhodamine auramine (Fig. 18B).

The M. tuberculosis strains revealed phenotype-differences in case of pellicle formation. The Δupk mutant was hampered in pellicle formation and the complementation strain developed a [page 63↓]gapless, smooth pellicle in contrast to the rough pellicle of M. tuberculosis wildtype (Fig. 18C).

Cultures of M. tuberculosis H37Rv and the Δupk mutant were grown to late log-phase (OD600 = 1.0) and whole cell lysates, as well as RNA were purified.

Proteome analysis was performed with 3 independent protein lysates of both strains, M. tuberculosis wildtype and M. tuberculosisΔupk mutant. Each sample was analyzed on 2 gels. The comparative proteome analysis of 12 gels (6 wildtype and 6 mutant) revealed 45 spots of differential relative intensity. For the Δupk mutant, 23 spots belonged to the group of higher intensity and 22 spots exhibited lower intensity (Table 2) compared to M. tuberculosis wildtype. Representative examples are depicted in Figure 19. The 2-DE gels were first evaluated visually. Subsequently, the gels were re-evaluated by means of the image analysis program PDQuest. This software allowed verification of significance of the results by t-test and offered the possibility to quantify spot intensities.

In parallel to determine and compare gene expression profiles, RNA of M. tuberculosis H37Rv and the Δupk strain were purified and hybridized to a M. tuberculosis array. Wildtype and mutant showed a high number of differentially regulated genes. The 20 most significantly regulated genes of 2 independent experiments are summarized in Figures 20 and 21.

Correlation of the results of proteome and transcriptome was limited. Comparing M. tuberculosisΔupk and M. tuberculosis wildtype, proteins of higher spot intensity, such as [page 65↓]Rv0341 and Rv2462 had a higher transcription rate, too. And proteins of lower spot intensity, like Rv1912 and Rv0125 also had a lower transcription rate. However, Rv2031c / HspX was a protein of high abundance in M. tuberculosisΔupk whole cell lysates, compared to M. tuberculosis wildtype, but gene expression did not differ between both strains. HspX is of interest, because it is one of several proteins produced during persistence of bacteria under low oxygen conditions and within granulomas, but at a lower level during logarithmic growth phase [64,65]. The examined bacterial cultures were harvested at late logarithmic growth phase (OD600 = 0.8 - 1).

Fig. 19 Examples of protein spots with differential relative intensity in 2-DE patterns of whole cell lysate proteins of the upk mutant strain (B, D, F) and the M. tuberculosis H37Rv control (A, C, E). The same protein can give rise to multiple spots as in the case of Rv2031c/HspX (picture E).

Thirty percent of the genes with a higher transcription rate in M. tuberculosisΔupk mutant compared to M. tuberculosis wildtype encode for proteins involved in lipid metabolism, additional 10 % were cell wall and cell-process related genes. Taken together, 40 % of the most significantly up-regulated genes were involved in the cell envelope complex. This corresponds to the observation that within the group of higher intensity protein spots, 37 % [page 75↓]belong to the cell envelope complex.

Twenty percent of the genes with a lower transcription rate in M. tuberculosisΔupk encode for proteins which are related to the cell envelope complex. Again, this corresponds to 21 % of the protein spots with lower intensity which are related to cell envelope complex.

Fig. 20 Percentage distribution of gene expression profiles and differential protein spot intensities according totuberculist classification of functional categories. Lipid metabolism was the most prominent fraction within the group of up-regulated genes for the Δupk mutant (A) and insertion sequences and phages protrude as a cluster of genes to be turned off (B).

[page 76↓]Global analysis pointed to especially one significantly upregulated gene cluster of transcriptome analysis: fabD (rv2243), kasA (rv2245), and accD6 (rv2247), and additionally from proteome analysis: KasB (Rv2246). This represents 4 members of a 5 gene operon (Fig. 21), consisting of FAS-II system encoding enzymes involved in biosynthetic pathway for long-chain fatty acids and precursors of mycolic acids which are part of MAPc. The higher transcription rate of a FAS-II system related operon in the case of the Δupk deletion mutant may reflect a mechanism to overcome the upk deficiency. The rv2244 gene and its geneproduct were inconspicuous concerning transcriptome and proteome analysis.

The alamar blue assay was used to determine sensitivity to antibiotics in the same way as described for M. smegmatis. Since there was no access to an Elisa-reader under biosafety level 3 (BSL3) conditions, completed assays were documented with a digital camera and visual color change was the value of bacterial growth. Alamar blue turns from blue to red upon reduction, indicating growth. Based on observations with M. smegmatis, the Δupk mutant was expected to be more sensitive to bacitracin. In contrast, no difference between M. tuberculosis H37Rv wildtype and Δupk mutant was detectable. The reconstituted strain was slightly more susceptible to bacitracin at minute concentration differences of the antibiotic. M. tuberculosis H37Rv and M. tuberculosisΔupk were resistant to bacitracin up to a concentration of 20 units / ml (Fig. 20A).

KasA and KasB which are over-expressed in the M. tuberculosisΔupk mutant, had been proposed to contribute to Isoniazid-resistance [66,67]. Sensitivity of the Δupk mutant to this antibiotic was therefore determined. All three examined M. tuberculosis strains were able to grow at concentrations of up to 20 µg Isoniazid / ml (Fig. 22) except for M. tuberculosisΔupk mutant which was slightly more susceptible, rather contradicting profound contribution of KasA to Isoniazid resistance.

To determine whether the upk deletion influenced growth of the mutant in vivo, C57BL/6 mice were infected intranasally with 1x103 cfu. Five mice per group, infected with M. tuberculosis H37Rv, M. tuberculosisΔupk, and M. tuberculosisΔupk + pMV262-upk, were sacrificed at day 1 to precisely determine the concentration of the inoculum. At day 30, 60, and 90, bacterial load in lung and spleen were determined (Fig. 23). M. tuberculosis H37Rv showed a characteristic growth curve with a plateau in the lung at 1x107 cfu. In contrast, cfu of the Δupk mutant increased less than a log per lung during the first 30 days and remained at a level of about 1.4 x 104 cfu / lung until day 90. The reconstituted strain reached an almost constant bacterial load of 1.8 x 103 cfu / lung at day 90. Nevertheless, all 3 strains were able to disseminate and were detected in spleen and liver (data not shown). While the burden of wildtype bacteria in spleen rose up to 1 x 105 cfu / organ by day 60, the burden of the upk mutant did exceed 4.1 x 103 cfu / spleen at day 30 and decreased afterwards. The phenotype of the reconstituted strain was unexpected and exhibited a slightly lower bacterial load than the M. tuberculosisΔupk strain in lung and spleen.

Organs of infected animals, were removed for histological examinations. At day 90 post infection, lungs of M. tuberculosis wildtype and M. tuberculosisΔupk mutant infected mice revealed striking differences. Typically, animals infected with M. tuberculosis H37Rv showed severe pathology in the lung (Fig. 24). Granulomas were formed and a major part of the lung consisted of affected tissue. In the case of upk deficient M. tuberculosis(Fig. 25), granuloma and affected tissue were found but, in contrast to infection with M. tuberculosiswildtype, the majority of the lung appeared unaffected. The same observation was made for the reconstituted strain which failed to mimic the characteristics of the wildtype strain (Fig. 26).

Fig. 24 Lungs of mice infected with M. tuberculosis H37Rv exhibited severe pathology 90 days post infection. Major part of the lung consisted of granulomatous, heavily infiltrated tissue. Only small regions with unaffected alveoli were found.

Fig. 25 Lungs of mice infected with M. tuberculosis H37Rv Δupk exhibited reduced pathology compared to M. tuberculosis wildtype 90 days post infection. Major part of the lung appeared non-infiltrated. Only small parts with granuloma could be detected.

Fig. 26 Lungs of mice infected with the reconstitution strain of M. tuberculosis H37Rv Δupk exhibited – like M. tuberculosisΔupk - reduced pathology compared to M. tuberculosis wildtype 90 days post infection. Major part of the lung appeared non-infiltrated. Only small parts with granuloma could be detected.

Knowledge about virulence of a pathogen can be acquired by infection of immunocompromised mice which fail to control disease beyond a certain threshold. Commonly used immunocompromised animal models are i) highly susceptible Rag1-/- mice which lack T and B cells and hence, fail to mount an adaptive immune response [68] and ii) IFNγ-/- mice which are deficient in generating the central mediator of protection against tuberculosis. In this study, Rag1-/- mice were infected with 1x106 cfu mycobacteria. M. tuberculosis wildtype infected mice survived 26 days in the median (range: d23 - d49). Mice infected with M. tuberculosisΔupk survived 70 days in the median (range: d67 - d77) . Survival times of wildtype and mutant infected animals were significantly different according to logrank test (P<0.0001). The reconstituted strain killed mice after day 42 and about 50 % of the mice survived longer than those infected with the Δupk strain (Fig. 27A).

Similar to Rag1-/- mice, IFNγ-/- animals were less susceptible to the Δupk mutant strain than to M. tuberculosis H37Rv wildtype. The median survival time of wildtype infected animals was 30 days (range: d26 – d32), and for M. tuberculosisΔupk mutant infected animals, 80 days (range: d63 – d126). Survival times of wildtype and mutant infected animals were significantly different according to logrank test (P<0.0001). Unexpectedly, the reconstituted strain was more attenuated than the mutant (Fig. 27B).

A upk deletion mutant (M. tuberculosisΔupk) was generated on a M. tuberculosis H37Rv background. In vitro growth properties of the mutant in shaking culture in 7H9 complete medium, cording, and neutral red to staining of this strain were unaffected. In addition, standing cultures in Sauton medium exhibited impaired pellicle formation. Global analysis of proteome and transcriptome revealed a high number of genes associated with lipid metabolism upregulated in the mutant, especially an operon of the mycobacterial FAS-II system. Increased sensitivity to bacitracin, however, was not detected. The upk deficient M. tuberculosis strain exhibited markedly reduced growth and pathology in vivo as well as reduced virulence in Rag1-/- and IFNγ-/- KO mice infections.

Balb/c mice were infected with 5 x 105 cfu of the M. bovis BCG Δupk strain and the M. bovis BCG wildtype strain i.v. and bacterial numbers were determined at days 15, 30, 60, and 90 post infection in lung and liver (Fig. 28). In the median, the bacterial burden in the lung of M. bovis BCG wildtype infected mice was about 2.17 times higher compared to mice infected with the upk deletion mutant, and 8.2 times higher in the liver. The small difference in bacterial load remained almost constant over the observed period of 90 days but was not significant according to “Mann Whitney” test (lung P = 0.5; liver P = 0.2).

IFNγ production by spleen cells of infected and of non infected mice was addressed by ELISA-assay (Fig. 29). Cells of whole spleens were either stimulated or left unstimulated with M. tuberculosis protein extracts. Spleen cells derived from naive mice failed to produce IFNγ, whilst cells of M. bovis BCG wildtype infected mice produced high levels of IFNγ at day 60 and day 90 post infection. Delayed IFNγ production of a comparably high level was measured at day 90 post infection in the case of spleen cells derived from mice infected with the BCG Δupk deletion mutant.

Fig. 29 Measurement of IFNγ production by ELISA. Spleen cells of M. bovis BCG inoculated mice produced high levels of IFNγ upon stimulation at day 60 and 90 post infection. Within each group, left column displays unstimulated, right column displays stimulated samples. Spleen cells of mice vaccinated with upk deficient M. bovis BCG strain produced a delayed but also high response at day 90 post infection.

A major goal of studies with recombinant M. bovis BCG would be their utilization as improved vaccines. To exploit this possibility we compared vaccine efficacy of M. bovis BCG Δupk to the existing vaccine M. bovis BCG. Balb/c mice were vaccinated i.v. with 5 x 105 cfu. After 120 days these animals and an unvaccinated control group were challenged with about 200 cfu M. tuberculosis H37Rv delivered as aerosol. At distinct time-points thereafter, bacterial loads in lung and spleen were determined (Fig. 30). M. tuberculosis cfu differences in the spleen were very small between the groups 30, 60, and 120 days post infection. The M. tuberculosis burden in the lung of both groups of vaccinated animals was about the same and 60 fold lower compared to unvaccinated animals at day 30 post infection. However, by day 120 post infection, the BCG Δupk mutant strain was able to induce a significantly superior protection (Δlog = 2.7 in relation to naive mice) compared to wildtype M. bovis BCG (Δlog = 0.9) in the lung.